Eastern (EEEV), Venezuelan (VEEV), and Western (WEEV) equine encephalitis viruses are mosquito-transmitted alphaviruses with the potential to cause fatal neuroinvasive disease in humans and domesticated animals. These viruses also can be spread by the aerosol route and thus, have significant potential as bioterrorist agents. Currently, there are no antiviral agents approved for alphaviruses. The existing investigational live-attenuated VEEV vaccine (LAV; TC-83) was generated >40 years ago, is highly reactogenic, poorly immunogenic, and causes disease in up to 20% of recipients. For EEEV and WEEV, investigational formalin-inactivated vaccines have been given to at-risk workers. While both elicit antibody responses, they provide uncertain protection, require multiple booster injections, and in fact, the WEEV vaccine is no longer available. Here, we propose to develop a safe and effective LAV vaccine against EEEV using recent advances in understanding of the virulence mechanisms of the virus. These advances have allowed mutagenesis of EEEV that specifically reduces neurovirulence and increases immunogenicity. We plan to combine mutations to create an optimal vaccine strain and we propose this strategy as a platform for LAV versus other alphaviruses. We will identify correlates of protection against EEEV and address the extent of cross- protection against VEEV. The development of safe and protective vaccines against the encephalitic alphaviruses will provide an immediate counter-measure against disease emergence or bioterrorist introduction. For the first time with alphaviruses, the individua and combined effects of mutations with known mechanisms of action will be tested for effects on virus replication, disease and immunogenicity in animals.